The Center for Transcriptional Consequences of Human Genetic Variation (CTCHGV) will develop innovative and powerful genetic engineering methods and use them to identify genetic variations that causally control gene transcription levels. Genome Wide Association Studies (GWAS) find many variations associated with disease and other phenotypes, but the variations that may actually cause these conditions are hard to identify because nearby variations in the same haplotype blocks consistently co-occur with them in human populations, so that specifically causative ones cannot be distinguished. About 95% of GWAS variations are not in gene coding regions, and many of these presumably associate with altered gene expression levels. CTCHGV will identify the variations that directly control gene expression by engineering precise combinations of changes to gene regulatory regions that break down the haplotype blocks, allowing each variations'effect on gene expression to be discerned independently of the others. To perform this analysis, CTCHGV will extract ~100kbps gene regulatory regions from human cell samples, create precise variations in them in E. coli, and re-introduce the altered regions back into human cells, using zinc finger nucleases (ZFNs) to efficiently induce recombination. CTCHGV will target 1000 genes for this analysis (Aim 1), and will use human induced Pluripotent Stem cells (iPS) to study the effects of variations in diverse human cell types (Aim 2). To explore the effects of variations in complex human tissues, CTCHGV will develop methods of measuring gene expression at transcriptome-wide levels in many single cells, including in situ in structured tissues (Aim 3). Finally, CTCHGV will develop novel advanced technologies that integrate DNA sequencing and synthesis to construct thousands of large DNA constructs from oligonucleotides, that enable very precise targeting and highly efficient performance of ZFNs, and that enable cells to be sorted on the basis of morphology as well as fluorescence and labeling (Aim 4). CTCHGV will also develop direct oligo-mediated engineering of human cells, and create "marked allele" iPS that will enable easy ascertainment of complete exon distributions for many pairs of gene alleles in many cell types. RELEVANCE: CTCHGV methods will yield precise knowledge of effects of human genetic variations on gene expression that will both refine and go beyond GWAS-derived associations between non-coding variations and disease. Powerful new CTCHGV genetic engineering methods will directly enable gene therapy. CTCHGV iPS and single-cell transcriptome technologies will increase understanding of diverse and complex human tissues.